Method for treatment of cancer and infectious disease

ABSTRACT

In a method of treating a viral infection involving activation of NK cells, the improvement comprising administering to a patient receiving such treatment an effective NK-cell-activity facilitating amount of a histamine H 2  or 5-HT 1A  receptor agonist.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/574,874, filed on May 19, 2000, now U.S. Pat. No. ______,which is a continuation of U.S. patent application Ser. No. 09/465,432,filed on Dec. 21, 1999, now U.S. Pat. No. 6,155,266, which is acontinuation of U.S. patent application Ser. No. 09/033,110, filed Mar.2, 1998, now U.S. Pat. No. 6,000,516, which is a continuation of U.S.patent application Ser. No. 08/374,787, filed May 8, 1995, now U.S. Pat.No. 5,728,378.

FIELD OF THE INVENTION

[0002] The present invention concerns a pharmaceutical preparation orsystem for activation of natural killer cells (NK-cells), in order, forexample, to treat tumors or virus infections.

BACKGROUND OF THE INVENTION

[0003] Natural killer cells (NK-cells) are a group of spontaneouslycytotoxic lymphocytes that destroy tumor cells by lysis with no antigenspecificity or restriction by histocompatibility molecules. Monocytesare involved in the regulation of the NK-cell's function, both throughmechanisms involving cell contact and through providing soluble NKcell-regulating mediators. Recently, a cell contact-mediated mechanismhas been described whereby monocytes regulate NK-cells. This type ofmonocyte-mediated regulation is exerted by monocytes that are obtaineddirectly from peripheral blood through counterflow centrifugalelutriation (CCE) and is regulated by the biogenic amines histamine andserotonin (Hellstrand and Hermodsson, 1986, J. Immunol. 137, 656-660;Hellstrand and Hermodsson, 1987, J. Immunol. 139, 869-875; Hellstrandand Hermodsson, 1990, Scand. J. Immunol. 31, 631-645; Hellstrand andHermodsson, 1990, Cell. Immunol. 127, 199-214; Hellstrand, Kjellson andHerrnodsson, 1991, Cell. Immunol., 138, 44-54). These NK-cell regulatingmechanisms caused by biogenic amines should be of importance to theNK-cell-mediated defense against metastatic tumors in vivo (Hellstrand,Asea and Hermodsson (1990), J. Immunology 145, 4365-4370).

[0004] Interferon-α (IFN-α) is an important regulating factor for NKcells. It effectively enhances the NK cell's cytotoxicity (NKCC) both invivo and in vitro (Trinchieri, 1989, Adv. Immunol. 47,187-376; Einhorn,Blomgren and Strander, 1978, Int. J. Cancer 22, 405-412; Friedman andVogel, 1984, Adv. Immunol., 34, 97-140).

[0005] Owing to the high rate of cancer and the only partiallysuccessful treatment methods available today, there is a constant demandfor other improved methods of treatment of tumors. There is also a greatdemand for improved treatment methods for virus infections.

SUMMARY OF THE INVENTION

[0006] The goal of the invention is to create a pharmaceuticalpreparation or system that effectively stimulates NK cells, e.g., inorder to treat tumors, primarily myelomas, renal cancer, leukemias andmelanoma, or to treat virus infections, primarily chronic hepatitis Band hepatitis C. The preparation or system according to the inventioninvolves a first composition, containing interferon-α or analoguesthereof, and a second composition containing at least one substance withhistamine H₂, or serotonin 5-HT_(1A) receptor agonist activity, wherebysaid first and second compositions are either mixed in a preparation orsupplied in separate doses in an amount sufficient for the intendedtreatment. The invention also comprises a method for treatment of viralor neoplastic disease comprising the step of coadministeringinterferon-α and an effective amount of a histamine H₂ receptor agonistor a serotonin 5-HT_(1A) receptor agonist. Furthermore, the inventionincludes a method for the treatment of viral infection comprisingadministering a histamine H₂ receptor agonist or a 5-HT_(1A) receptoragonist.

[0007] The invention shall be described in greater detail below, makingreference to reported in vitro experiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008]FIG. 1 shows in graph form the synergistic NK cell activationagainst cultured target cells produced by IFN-α and histamine orserotonin for various concentrations of IFN-α (0- 100 U/ml).

[0009]FIG. 2 shows in graph form the synergistic NK cell activationproduced against freshly recovered human leukemic cells by IFN-α andhistamine for various concentrations of IFN-α (0-100 U/ml).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The invention is based on the unexpected discovery that IFN-α andthe biogenic amines histamine and/or serotonin produce a synergisticactivation of NK cells.

[0011] The experiments reported hereafter show that eluted monocyteseffectively suppress the activation of NK cells induced by IFN-α.Furthermore, it is shown that histamine or serotonin, which act throughdefined bioaminergic receptors, remove the monocyte induced suppressionand thereby restore the ability of the NK cells to respond to IFN-α.

[0012] Analogues of histamine with H2-receptor agonist activity or othercompounds with H2-receptor agonist activity and analogues of serotoninwith 5-HT_(1A)-receptor agonist activity or other compounds with5-HT_(1A)-receptor agonist activity that are suitable for use in thepresent invention are known within the art and shall not be describedmore closely here. For example, these analogues can have a chemicalstructure resembling that of histamine or serotonin, but modified byaddition of groups that do not negatively affect the H₂ or 5-HT_(1A)receptor activities. Known H₂-receptor agonists include histamine,dimaprit, clonidine, tolazoline, impromadine, 4-methylhistamine,betazole and histamine congener derivatives such as:

[0013] described as compounds 1, 6, and 9 in Khan et al., J. Immunol.,Vol. 137 pp. 308-315. Known serotonin 5-HT_(1A) receptor agonistsinclude 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), ALK-3(cis-8-hydroxy-1-methyl-2-(di-n-propylamino)tetralin), BMY 7378(8[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione),NAN 190 (1-(2-methoxyphenyl-4-[4-(2-phthalimmido)butyl]pierazine HBr),lisuride, d-LSD, flesoxinan, DHE (dihydroergotamine), MDL 72832(8-[4-91,4-benzodioxan-2-ylmethyl-amino)butyl]-8-azaspiro[4,5]decane-7,9-dione), 5-CT (5-carboxamidotryptamine), DP-5-CT(N,N-dipropyl-5-carboxamidotryptamine), ipsapirone, WB 4101(2-[[[2-(2,6-dimethoxyphenoxy)ethyl]amino]methyl]-1,4-benzodioxane),ergotamine, buspirone, metergoline, spiroxatrine, PAPP(1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl) piperazine), SDZ(−) 21009((4(3-terbutylamino-2-hydroxypropoxy)indol-2-carbonic-acidisopropylester),and butotenine.

[0014] IFN-α and histamine/serotonin can be administered separately orin the same preparation. The method of administration can be eitherlocal or systemic injection or infusion. Other methods of administrationcan also be suitable.

[0015] The compounds can even be administered intraperitoneally or inanother parenteral method. Solutions of the active compounds in the formof free acids or pharmaceutically acceptable salts can be administeredin water with or without a tenside such as hydroxypropylcellulose.Dispersions making use of glycerol, liquid polyethyleneglycols, ormixtures thereof with oils can be used. Antimicrobial compounds can alsobe added to the preparation.

[0016] Injectable preparations may include sterile water-based solutionsor dispersions and powders that can be dissolved or suspended in asterile medium prior to use. Carriers such as solvents or dispersantscontaining, e.g., water, ethanolpolyols, vegetable oils and the like canalso be added. Coatings such as lecithin and tensides can be used tomaintain suitable fluidity of the preparation. Isotonic substances suchas sugar or sodium chloride can also be added, as well as productsintended to retard absorption of the active ingredients, such asaluminum monostearate and gelatin. Sterile injectable solutions areprepared in the familiar way and filtered before storage and/oradministration. Sterile powders can be vacuum-dried or freeze-dried froma solution or suspension.

[0017] All substances added to the preparation must be pharmaceuticallyacceptable and essentially nontoxic in the quantities used. Thepreparation and formulations that produce a delayed release are alsopart of the invention.

[0018] The preparation is supplied in dosage units for a uniform dosageand to facilitate administration. Each dosage unit contains apredetermined quantity of active components to produce the desiredtherapeutic effect, along with the requisite quantity of pharmaceuticalcarriers.

[0019] IFN-α can be administered in a quantity of around 1000 to 300,000U/kg/day, preferably around 3000 to 100,000 U/kg/day and morepreferably, around 10,000 to 50,000 U/kg/day.

[0020] The compounds with H₂, and 5-HT_(1A) receptor agonist activitycan be administered in a of quantity of around 0.1 to 10 mg/day,preferably around 0.5 to 8 mg/day and more preferably, around 1 to 5mg/day. However other quantities can be administered with IFN-α, asdecided by the treating physician. For substances other than biogenicamines with corresponding receptor activity, doses producing anequivalent pharmacological effect shall be used.

[0021] Although it is stated in the examples that the administration wasgiven in a single dose, it is obvious that the compounds can bedistributed over longer periods of time for treatment of virusinfections or tumors.

[0022] The daily dose can be administered as a single dose or it can bedivided into several doses, should negative effects occur.

EXAMPLES In Vitro Studies of IFN-α and Histamine/Serotonin

[0023] This example illustrates the effect of human recombinant IFN-αand histamine/serotonin, separately and in combination, on the NK cellcytotoxicity (NKCC) for human mononuclear cells (MNC).

[0024] MNC were obtained from peripheral venous blood from healthy humanblood donors by Ficoll-Hypaque centrifuging, followed by Percolldensity-gradient fractionation (Timonen and Saksela, 1980, J. Immunol.Methods 36, 285-291; Hellstrand and Hermodsson, 1990, Scand. J. Immunol.31, 631-645).

[0025] In the respective Percoll fractions, the high-density MNC(Percoll fractions 1-4) were small lymphocytes with low baselinecytotoxicity against K562 target cells. After removal of the monocytes,the low-density fractions 6-10 displayed high NKCC, consistent withearlier studies. (Timonen and Saksela, 1980, J. Immunol. Methods 36,285-291).

[0026] The target cells used in these experiments were K562, an NK-cellsensitive erythroleukemic cell line, or Daudi, a relativelyNK-insensitive EBV-transformed B-cell lymphoblastoid cell line.

[0027] The NKCC was determined six times as the specific ⁵¹Cr-releasefor a NMNC: target-cell ratio of between 30:1 and 3.8:1 in two-folddilution gradients. The suspensions of MNC/target cells were incubatedin microplates at 37° C. for 6 hours (Daudi) or 16 hours (K562). Thesupernatant solution was then collected and examined for radioactivityin a gamma counter. The maximum ⁵¹Cr-release was measured in target cellcultures treated with Triton X-100. The NKCC was calculated as the celllysis % by the formula 100×(experimental release−spontaneousrelease/maximum release−spontaneous release)=cell lysis %.

[0028] A low-density Percoll fraction was separated by counterflowcentrifuge elusion (CCE) in a monocyte and in a lymphocyte fraction. Themonocyte fraction was concentrated to >90% purity whereupon thecontaminating cells consisted of large lymphocytes. The lymphocytefractions obtained by CCE contained <3% monocytes, determined bymorphology and Leu-M3 (CD14) antigen expression. The lymphocytes wereCD3⁻/16⁺/56⁺ T cells (45-50%), CD3⁻/16⁻/56⁻NK cells (35-40%) ,CD3⁺/16⁻/56⁻ T cells (45-50%), CD3⁺/16⁺/56⁺ cells (1-5%), determined byflow cytometry.

[0029] J The eluted monocytes and/or the NK cell-concentratedlow-density lymphocytes were treated with IFN-α and histamine/serotonin.The compounds were added, separately or in combination, to mixtures ofMNC and K562 target cells at the start of a 16-hour ⁵¹Cr-release assay.The cytotoxicity against K562 in the NK cell-concentrated lymphocytefraction was increased by IFN-α and unaffected by histamine orserotonin. The eluted monocyte fraction exhibited a low baselinecytotoxicity and was slightly induced by histamine/ IFN-α or serotonin/IFN-α; this cytotoxicity resulted from the low fraction of contaminatinglymphocytes (data not given). The addition of eluted monocytes to the NKcell concentrated lymphocytes suppressed the baseline cytotoxicity toK562. Furthermore, the eluted monocytes almost totally inhibited theactivation of the cytotoxicity by means of IFN-α (Table 1) .

[0030] Histamine and serotonin restored the basal cytotoxicity oflymphocytes in mixtures of monocytes and lymphocytes. Furthermore, bothhistamine and serotonin eliminated the monocyte induced inhibition ofthe NK cell response to IFN-α. Hence, IFN-α plus histamine or serotoninsynergistically enhance the cytotoxicity in mixtures of monocytes and NKcell-enriched lymphocytes (Table 1).

[0031] In the experiments reported in Table 1, eluted lymphocytes weremixed with monocytes as shown in the table, in a total volume of 150 μl.The data are NKCC (mean ±SEM) of six determinations. Serotonin 10⁻⁴ Mand/or IFN-α (25 U/ml) was added at the start of a 16-hourmicrocytotoxicity test against 10⁴ K562 target cells. TABLE 1Suppression of NK Cell Cytotoxicity by Monocytes and Elimination of ThisEffect with Serotonin NK CELL CYTOTOXICITY AFTER TREATMENT WITH (×10⁻⁴)Lymphocytes (×10⁻⁴) Control Serotonin IFN Serotonin + IFN 0 12 34 ± 1 34± 3 58 ± 3 60 ± 2 6 12 10 ± 2 31 ± 2 17 ± 1 52 ± 2 12 12  9 ± 1 31 ± 210 ± 1 52 ± 2

[0032] Table 2 shows the synergistic activation of NK cells by combinedtreatment with IFN-α and histamine. Monocytes were recovered along withNK cells in low-density Percoll fractions. In the experiment shown inTable 2, IFN-α and/or histamine was added to MNC obtained from thesemonocyte-containing Percoll fractions. As was the case with mixtures ofeluted monocytes and low-density lymphocytes, IFN-α was relativelyineffective in these cell fractions, while histamine increased thecytotoxicity. Treatment of monocyte-containing cells with histamine(10⁻⁴ - 10⁻⁶ M) and IFN-α (25 U/ml) produced a synergistic NK-boostingresponse against K562 and against Daudi target cells. A similar resultwas obtained when histamine was replaced by serotonin.

[0033] In the results shown in Table 2, MNC from five different donorswere used. All compounds were added to mixtures of MNC and target cellsat the start of a 6 h (Daudi) or 16 h (K562) effecter and target cellincubation. The effecter cells were obtained from Percoll fractions 7-8,containing 33-55% monocytes.

[0034]FIG. 1 shows the synergistic NK cell activation by IFN-α andhistamine/serotonin for different concentrations of IFN-α (0-100 U/ml).Cells from the monocyte-containing Percoll fraction 8 were incubatedwith culture medium, histamine (10⁻⁴ M) or serotonin (10⁻⁴ M) in thepresence of IFN-α (0-100 U/ml). The data shown are NKCC (cell lysis %;mean±SEM of six determinations). The compounds were added at the startof a 16 h microcytotoxicity test against K 562 target cells. TABLE 2Synergistic Activation of NK Cells by Histamine and IFN-α MNC/ targetNKCC (cell lysis % ± SEM) Target cell Histamine concentration Exp cellratio Treatment 0 10⁻⁴ M 10⁻⁵ M 10⁻⁶ M 1 K562 15:1 Medium 33.1 ± 0.555.5 ± 1 54.7 ± 1 39.2 ± 1 IFN 25 U/ml 33.1 ± 1   76.4 ± 3 74.1 ± 1 66.0± 2 2 K562 15:1 Medium 20.7 ± 0.4 32.4 ± 1 27.4 ± 1 23.2 ± 2 IFN 25 U/ml27.4 ± 1   67.9 ± 2 66.2 ± 1 55.4 ± 1 3 K562 15:1 Medium 31.4 ± 1   43.3± 1 38.6 ± 1 29.4 ± 1 IFN 25 U/ml 32.5 ± 1   71.9 ± 1 66.5 ± 2 56.3 ± 24 Daudi 30:1 Medium  1.0 ± 0.4  4.4 ± 1  3.5 ± 2   1.1 ± 0.3 IFN 25 U/ml 1.1 ± 0.5 31.7 ± 1 28.3 ± 1 14.1 ± 1 5 Daudi 30:1 Medium 2.2 ± 1  13.5± 1  9.7 ± 1  2.5 ± 1 IFN 25 U/ml 2.7 ± 1  61.3 ± 3 52.3 ± 2 31.7 ± 1

[0035] The effect of histamine on monocyte-induced suppression ofresting and IFN-α-activated NK cells was completely blocked bysimultaneous treatment with the specific H₂R antagonist ranitidine andimitated by the H₂R agonist dimaprit, which is shown in Table 3. Thismeans that the effect of histamine on the NK cell's response to IFN-α isH₂R-specific. TABLE 3 Effects of Histamine and H₂R Agonist Dimaprit andH₂-Antagonist Ranitidine on NK Cells NKCC (Cell Lysis %) ± SEM AFTERTREATMENT WITH Treatment Control Ran IFN Ran + IFN Control 0.1 ± 0.1 0.0± 0.1  0.1 ± 0.1 0.0 ± 0.1 Histamine 9.4 ± 0.3 1.5 ± 0.3 31.7 ± 0.3 1.6± 0.2 Dimaprit 6.4 ± 1   0.4 ± 0.4 32.6 ± 1   0.5 ± 0.5

[0036] In the experiment shown in Table 3, culture medium (control),histamine (10⁻⁴ M), dimaprit (10⁻⁴ M), ranitidine (ran) (10⁻⁴ M) and/orIFN-α (25 U/ml) were added at the start of a 6-hour ⁵¹Cr release assayusing Daudi target cells. The data are representative of three similarexperiments. NKCC is given as mean cell lysis %±SEM of sixdeterminations. The effecter cells were recovered from a low-densityPercoll fraction 8, containing around 40% monocytes.

[0037] Serotonin acted synergistically with IFN-α and had an effectcorresponding to that of histamine. Ranitidine ( 10⁻⁴ M) did not alterthe effect of serotonin. The specific synthetic 5-HT_(1A) R-agonists8-OH-DPAT and (+) - ALK-3, which lack activity for 5-HT₁₈R; 5-HT₁₀R,5-HT₂R or —HT₃R, intensified the baseline NKCC and restored the NKcell's response to IFN-α with a potency and effect comparable to that ofserotonin. This is shown in Table 4. Ketanserin and ondansetron, whichare antagonists of 5-HT₂R and 5-HT₃R, respectively, did not influencethe effect of serotonin in equimolar concentrations. TABLE 4 The Effectof Serotonin and 5-HT_(1A)R Agonists on NK Cells NKCC AFTER TREATMENTWITH Treatment Medium IFN Medium 1.1 ± 1   0.5 ± 0.3 Serotonin 10⁻⁴ M10.4 ± 1  44.3 ± 1 Serotonin 10⁻⁵ M   4.5 ± 0.3 33.2 ± 1 Serotonin 10⁻⁶M   2.2 ± 0.4 12.3 ± 1 8-OH-DPAT 10⁻⁴ M 8.8 ± 1 43.3 ± 1 (+)-ALK-3 10⁻⁴M 9.1 ± 1 40.4 ± 1

[0038] In the experiment shown in Table 4, culture medium (control),serotonin, 8-OH-DPAT (+)-ALK and/or IFN-α (25 U/ml) were added at thestart of a 6-hour ⁵¹Cr-release assay against Daudi target cells. TheNKCC is given as cell lysis %±SEM of six determinations. The effectorcells were recovered from the low-density Percoll fraction 7, containingaround 36% monocytes.

[0039] Similar experiments were then performed using freshly recoveredhuman tumor cells as target cells, rather that the cultured tumor celllines used as target cells in the experiments described above.

[0040] MNC were obtained from peripheral venous blood by Ficoll-Hypaquecentrifuging and the mononuclear cells were separated into monocytes andNK-cell-enriched lymphocytes (Hellstrand et al., J. Interferon Res., 12,199-206 1992). Seventy thousand NK-cell-enriched lymphocytes were mixedwith 70,000 monocytes and 20,000 ⁵¹Cr-labeled leukemic target cells (97%pure acute myelogenous leukemic cells) in a total volume of 150 μl. Thecells were treated with culture medium (control) or histaminedihydrochloride at a final concentration of 10⁻⁴ M, during a 16 hour⁵¹Cr-release assay to determine killed target cells.

[0041] The results are shown in FIG. 2. The data are the mean percentcell lysis of six determinations ±SEM. The recorded cytotoxicity wascompletely depleted after removal of NK-cells using DYNABEADS coatedwith anti-CD56, but not by removal of T-cells using beads coated withanti-CD3 (Hellstrand et al., Scand. J. Immunol., 37:7-18 (1993). As seenin FIG. 2, treatment with interferon alone does not induce killing ofleukemic target cells unless histamine is present. In addition, it hasbeen shown that the cytotoxic effects obtained with histamine andinterferon-α are seen not only in cultured tumor cells, but in freshlyrecovered human leukemic cells as well.

[0042] Thus, in conclusion, it can be affumed that the above-describedin vitro experiments demonstrate that the biogenic amines histamine,through H₂ type receptors, and serotonin, through 5-HT_(1A) typereceptors, abolish the monocyte-induced suppression of resting and IFN-αactivated NK cells. Treatment with IFN-α and compounds with H₂ orHT_(1A) receptor agonist activity thus produces a synergistic activationof NK cells, which can be used in connection with tumor treatment ortreatment of virus infections.

What is claimed is:
 1. A method for the treatment of cancer in a patientthrough activation of NK cells, the improvement comprising administeringto the patient an effective NK-cell-activity facilitating amount of ahistamine H₂ or 5-HT_(1A) receptor agonist.
 2. The method of claim 1,wherein the receptor agonist is histamine.
 3. The method of claim 1,wherein the receptor agonist is a histamine congener.
 4. The method ofclaim 1, wherein the receptor agonist is serotonin.
 5. The method ofclaim 1, wherein the receptor agonist is selected from the groupconsisting of dimaprit, clonidine, tolazoline, impromadine,4-methylhistamine, betazole, 8-OH-DPAT(8-hydroxy-2-(di-n-propylamino)tetralin), ALK-3(cis-8-hydroxy-1-methyl-2-(di-n-propylamino)tetralin), BMY 7378(8[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione),NAN 190 (1-(2-methoxyphenyl-4-[4-(2-phthalimmido)butyl]pierazine HBr),lisuride, d-LSD, flesoxinan, DHE (dihydroergotamine), MDL 72832(8-[4-91,4-benzodioxan-2-ylmethyl-amino)butyl]-8-azaspiro[4,5]decane-7,9-dione), 5-CT (5-carboxamidotryptamine), DP-5-CT(N,N-dipropyl-5-carboxamidotryptamine), ipsapirone, WB 4101(2-[[[2-(2,6-dimethoxyphenoxy)ethyl]amino]methyl]-1,4-benzodioxane),ergotamine, buspirone, metergoline, spiroxatrine, PAPP(1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl) piperazine), SDZ(−) 21009((4(3-terbutylamino-2-hydroxypropoxy)indol-2-carbonic-acidisopropylester),and butotenine.
 6. The method of claim 1, wherein said receptor agonistis administered in a daily dose of between about 0.1 and 10 mg.
 7. Themethod of claim 1, wherein said cancer is a myeloma.
 8. The method ofclaim 1, wherein said cancer is a renal cancer.
 9. The method of claim1, wherein said cancer is a leukemia.
 10. The method of claim 1, whereinsaid cancer is a melanoma.